Methods and compositions for lipidization of hydrophilic molecules

ABSTRACT

Fatty acid derivatives of sulfhydryl-containing compounds (for example, sulfhydryl-containing peptides or proteins) comprising fatty acid-conjugated products with a disulfide linkage are employed for delivery of the compounds to mammalian cells. This modification markedly increases the absorption of the compounds by mammalian cells relative to the rate of absorption of the unconjugated compounds, as well as prolonging blood and tissue retention of the compounds. Moreover, the disulfide linkage in the conjugate is quite labile in the cells and thus facilitates intracellular release of the intact compounds from the fatty acid moieties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/120,118, filed Jul. 22, 1998, now U.S. Pat. No. 6,225,445, which is adivisional of U.S. application Ser. No. 08/524,362, filed Sep. 5, 1995,now U.S. Pat. No. 5,907,030, which is a continuation-in-part of U.S.application Ser. No. 08/349,717, filed Jan. 25, 1995, now abandoned.

This application is a continuation-in-part of application Ser. No.08/349,717 filed Jan. 25, 1995.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of biology andmedicine. More particularly, the present invention is directed tomethods and compositions useful in increasing in mammals the absorptionand retention of hydrophilic molecules, in particular peptides andproteins.

Advances in biotechnology have made possible the production of largeamounts of therapeutically active and pure proteins and peptides.Currently, the therapeutic effects of most of these agents can beachieved only when they administered via invasive routes, such as byinjection. Since most proteins have very short half lives, effectiveconcentrations of these agents can be maintained only when administeredby frequent injections.

Although the administration of proteins by injection is the mosteffective means of their delivery in vivo, patient tolerance of multipleinjections is very poor. In addition, the administration of drugs viathe injection routes is a skilled job and requires training; this skilland training may not always be transferable to patients. In cases whereprotein drugs have a life-saving role, the administration by theinjection route can be accepted by the patients. However, in cases whereprotein drugs are just one of several possible therapies, injections ofproteins and peptides are unlikely to be accepted by the patients.Therefore, alternative routes of protein and peptide delivery need to bedeveloped.

Alternative routes of protein and peptide delivery may include thebuccal, nasal, oral, pulmonary, rectal and ocular routes. Withoutexception, these routes are less effective than the parenteral routes ofadministration. However, these routes of protein and peptide deliveryare still far more attractive than the parenteral routes because theyoffer convenience and control to the patients. The oral route isparticularly attractive because it is the most convenient andpatient-compliant.

Mucosal barriers, which separate the inside of the body from the outside(e.g. GI, ocular, pulmonary, rectal and nasal mucosa), comprise a layerof tightly joined cell monolayers which strictly regulates the transportof molecules. Individual cells in barriers are joined by tight junctionswhich regulate entry into the intercellular space. Hence, the mucosa isat the first level a physical barrier, transport through which dependson either the transcellular or the paracellular pathways [Lee, V. H. L.(1988) CRC. Critical Rev. Ther. Drug Delivery Sys. 5, 69–97].

Paracellular transport through water filled tight junctions isrestricted to small molecules (MW<1 kDa) and is essentially a diffusionprocess driven by a concentration gradient across the mucosa [Lee(1988), supra; Artursson, P., and Magnusson, C. (1990) J. Pharm. Sci.79, 595–600]. The tight junctions comprise less than 0.5% of the totalsurface area of the mucosa [Gonzalez-Mariscal, L. M. et al. (1985) J.Membrane. Biol. 86, 113–125; Vetvicka, V., and Lubor, F. (1988) CRCCritical Rev. Ther. Drug Deliv. Sys. 5, 141–170]; therefore, they playonly a minor role in the transport of protein drugs across the mucosa.

The transcellular transport of small drugs occurs efficiently providedthe physiochemical properties of the drug are suited to transport acrosshydrophobic cell barriers. However, the transcellular transport ofproteins and peptides is restricted to the process of transcytosis[Shen, W. C. et al. (1992) Adv. Drug Delivery Rev. 8, 93–113].Transcytosis is a complex process in which proteins and peptides aretaken up into vesicles from one side of a cell, and are subsequentlyshuttled through the cell to other side of the cell, where they aredischarged from the endocytic vesicles [Mostov, K. E., and Semister, N.E. (1985) Cell 43, 389–390]. The cell membrane of mucosal barriers is ahydrophobic lipid bilayer which has no affinity for hydrophilic, chargedmacromolecules like proteins and peptides. In addition, mucosal cellsmay secrete mucin which can act as a barrier to the transport of manymacromolecules [Edwards, P. (1978) British Med. Bull. 34, 55–56].Therefore, unless specific transport mechanisms exist for protein andpeptide, their inherent transport across mucosal barriers is almostnegligible.

In addition to providing a tight physical barrier to the transport ofproteins and peptides, mucosal barriers possess enzymes which candegrade proteins and peptides before, after, and during their passageacross the mucosa. This barrier is referred to as the enzymatic barrier.The enzymatic barrier consists of endo- and exopeptidase enzymes whichcleave proteins and peptides at their terminals or within theirstructure. Enzymatic activity of several mucosa have been studied andthe results demonstrated that substantial protease activity exists inthe homogenates of buccal, nasal, rectal and vaginal mucosa of albinorabbits and that these activities are comparable to those present in theilium [Lee et al. (1988), supra]. Therefore, regardless of the mucosabeing considered, the enzymatic barrier present will feature strongly inthe degradation of the protein and peptide molecules.

The N and the C termini of peptides are charged and the presence ofcharged side chains impart highly hydrophilic characteristics on thesemacromolecules. In addition, the presence of charged side chains meansthat proteins and peptides have strong hydrogen binding capacities; thisH-binding capacity has been demonstrated to play a major role ininhibiting the transport of even small peptides across cell membranes[Conradi, R. A. et al. (1991) Pharm. Res. 8, 1453–1460]. Therefore, thesize and the hydrophilic nature of proteins and peptides combine toseverely restrict their transport across mucosal barriers.

One approach that has been used to alter the physical nature of themucosal barriers is the use of penetration enhancers. The use ofpenetration enhancers is based on the disruption of the cell barriers bythe use of low molecular weight agents which can fluidize cell membranes[Kaji, H. et al. (1985) Life Sci. 37, 523–530], open tight junctions[Inagaki, M. et al. (1985) Rhinology 23, 213–221], and create pores inthe cell membrane [Gordon, S. et al. (1985) Proc. Natl. Acad. Sci. USA82, 7419–7423; Lee, V. H. L. et al. (1991) Critical Reviews inTherapeutic Drug Carrier Systems, CRC Press 8, 91–192]. The use of theseagents leads to a non-specific loss of barrier integrity and can lead tothe absorption of a variety of large molecules which can be toxic tocells in vivo.

Protease inhibitors have been co-administered with proteins and peptidesand have shown some limited activity in enhancing the absorption ofthese macromolecules in vivo [Kidron, M. et al. (1982) Life Sci. 31,2837–2841; Takaroi, K. et al. (1986) Biochem. Biophys. Res. Comm. 137,682–687]. The safety and the long term effects of this approach have yetto be thoroughly investigated.

The prodrug approach is based on the modifications of peptides in amanner that will protect them from enzyme degradation and recognition.This has been achieved by substitution of the D-forms of amino acids inthe structure of peptides, the blockage of vulnerable groups on peptidesby amidation and acylation, the inversion of the chirality of peptides,and the introduction of conformational constraints in the peptidestructure. The synthesis of prodrugs is only applicable to smallpeptides which have easily identifiable domains of activity.

Reduction in size is another feasible approach to increasing thetransport potential of proteins. However, the active sites of proteinsneed to be mapped before size reduction can be attempted. In general,this approach is difficult to apply to the majority of proteins.

Carrier ligands, by virtue of their properties, can alter the celluptake and transport characteristics of proteins and peptides. Theessence of this approach is that a cell-impermeant protein or peptide iscovalently attached to a carrier which is highly transported into cells.The mechanisms through which carrier ligands become endocytosed andtranscytosed are important in deciding the suitability of the carrierfor enhancing the transport of proteins and peptides. Macromolecularcarriers are hydrophilic and do not partition into the membrane.Therefore, the transport of large polymeric carriers into the cells ismediated by the affinity of the carrier for the cell membrane.Generally, the uptake of a macromolecular conjugate starts with thebinding to the cell membrane. The binding of the carrier to the cellscan be specific (e.g. binding of antibodies to cell surface antigens),nonspecific (binding of cationic ligands or lectins to cell surfacesugars), or receptor mediated (binding of transferrin or insulin totheir receptors). Once the carrier is bound to the cell surface, it istaken up into vesicles. These vesicles then become processed stepwiseand can be routed to several pathways. One pathway is the recycling ofthe vesicle back to the membrane from which it was invaginated. Anotherpathway, which is destructive to the conjugate, is the fusion withlysosomes. An alternative pathway, and one which leads to thetranscytosis of the conjugate, is the fusion of the vesicle with themembrane opposite to the side from which it was derived.

The correct balance between the processes of endocytosis andtranscytosis determine the delivery of a protein conjugate to itstarget. For instance, endocytosis may determine the extent to which aconjugate is taken up by the target cell, but transcytosis determineswhether or not a conjugate reaches its target [Shen et al. (1992),supra]. For successful absorption through the GI-tract, a conjugate mustbind the apical membrane of the GI-mucosa, become internalized into themucosal cells, be delivered across the cells, and finally becomereleased from the basolateral membrane.

The current literature contains many reports which demonstrate thatnonspecific carriers, such as polylysines [Shen, W. C., and Ryser, H. J.P. (1981) Proc. Natl. Acad. Sci. USA 78 7589–7593] and lectins[Broadwell, R. D. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 632–646],and specific carriers, such as transferrin [Wan, J. et al. (1992) J.Biol. Chem. 267, 13446–13450], asialoglycoprotein [Seth, R. et al.(1993) J. Infect. Diseases 168, 994–999], and antibodies [Vitetta, E. S.(1990) J. Clin. Immunol. 10, 15S–18S] can enhance the endocytosis ofproteins into cells. Reports dealing with transcytotic carriers forproteins are fewer, and very few studies have quantitated the transportof protein conjugates across cell barriers. Wheat germ agglutinin[Broadwell et al. (1988), supra] and an anti-transferrin/methotrexateconjugate [Friden, P. M., and Walus, L. R. (1993) Adv. Exp. Med. Biol.331, 129–136] have been shown to be transcytosed across the blood brainbarrier in vivo. Also, polylysine conjugates of horseradish peroxidase(HRP) and a transferrin conjugate of HRP have been shown to betranscytosed across cell monolayers in vitro [Wan, J. and Shen, W. C.(1991) Pharm. Res. 8, S-5; Taub, M. E., and Shen, W. C. (1992) J. Cell.Physiol. 150, 283–290; Wan, J. et al. (1992) J. Biol. Chem. 267,13446–13450, supra].

Fatty acids, as constituents of phospholipids, make up the bulk of cellmembranes. They are available commercially and are relatively cheap. Dueto their lipidic nature, fatty acids can easily partition into andinteract with the cell membrane in a non-toxic way. Therefore, fattyacids represent potentially the most useful carrier ligands for thedelivery of proteins and peptides. Strategies that may use fatty acidsin the delivery of proteins and peptides include the covalentmodification of proteins and peptides and the use of fatty acidemulsions.

Some studies have reported the successful use of fatty acid emulsions todeliver peptide and proteins in vivo [Yoshikawa, H. et al. (1985) Pharm.Res. 2, 249–251; Fix, J. A. et al. Am. J. Physiol. 251, G332–G340]. Themechanism through which fatty acid emulsions may promote the absorptionof proteins and peptides is not yet known. Fatty acid emulsions may opentight junctions, solubilize membranes, disguise the proteins andpeptides from the GI environment, and carry proteins and peptides acrossthe GI-mucosa as part of their absorption [Smith, P. et al. (1992) Adv.Drug Delivery Rev. 8, 253–290]. The latter mechanism has been proposed,but is inconsistent with current knowledge about the mechanism of fatabsorption.

A more logical strategy to deliver proteins and peptides across theGI-epithelium is to make use of fatty acids as non-specific membraneadsorbing agents. Several studies have shown that a non-specificmembrane binding agent linked to a protein can promote the transcytosisof a protein conjugate across cells in vitro [Wan, J. et al. (1990) J.Cell. Physiol. 145, 9–15; Taub and Shen (1992), supra]. Fatty acidconjugation has also been demonstrated to improve the uptake ofmacromolecules into and across cell membranes [Letsinger, R. et al.(1989) Proc. Natl. Acad. Sci. USA 86, 6553–6556; Kabanov, A. et al.(1989) Protein Eng. 3, 39–42]. Nonetheless, there have been difficultiesin conjugating fatty acids to peptides and proteins, including: (1) thelack of solubility of fatty acids in the aqueous solution for theconjugation reaction; (2) the loss of biological activity of peptidesand proteins after fatty acid acylation; and (3) the lack of solubilittyof fatty acid-conjugated peptides in aqueous solutions [see, e.g.,Hashimoto, M. et al., Pharm. Res. 6, 171–176 (1989); Martins, M. B. F.et al., Biochimie 72, 671–675 (1990); Muranishi, S. et al., Pharm. Res.8, 649–652 (1991); Robert, S. et al., Biochem. Biophys. Res. Commun.196, 447–454 (1993)].

It is an object of the present invention to provide methods andcompositions for use in conjugating fatty acids to hydrophilic moleculesand in improving the bioavailability of peptides and proteins.

SUMMARY OF THE INVENTION

In accordance with the present invention, fatty acid derivatives ofsulfhydryl-containing compounds (for example, peptides, proteins oroligonucleotides which contain or are modified to contain sulfhydrylgroups) comprising fatty acid-conjugated products with a disulfidelinkage are employed for delivery of the sulfhydryl-containing compoundsto mammalian cells. This modification markedly increases the absorptionof the compounds by mammalian cells relative to the rate of absorptionof the unconjugated compounds, as well as prolonging blood and tissueretention of the compounds. Moreover, the disulfide linkage in theconjugate is quite labile in the cells and thus facilitatesintracellular release of the intact compounds from the fatty acidmoieties. Reagents and methods for preparation of the fatty acidderivatives are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Pursuant to the present invention, a sulfhydryl-containing compound (forexample, a biopolymer as hereinafter defined) is attached to a fattyacid derivative via a reversible, biodegradable disulfide bond. Such aconjugate would be expected to bind to the apical side of a cellmembrane, reach the basolateral membrane of the GI-epithelium as aresult of membrane transport and turnover, and become released intointerstitial fluid as the result of disulfide bond reduction.

Pursuant to one aspect of the present invention, there are providedconjugates of the general formula VI

in which P is a residue derived from a sulfhydryl-containing compound;R¹ is hydrogen, lower alkyl or aryl; R² is a lipid-containing moiety (ashereinafter defined); and R³ is —OH, a lipid-containing moiety or anamino acid chain comprising one or 2 amino acids and terminating in—CO₂H or —COR². These conjugates are particularly useful for increasingthe absorption and prolonging blood and tissue retention of thesulfhydryl-containing compound PSH.

Pursuant to another aspect of the present invention, methods forincreasing the absorption or prolonging blood and tissue retention in amammal of a sulfhydryl-containing compound of the general formula PSHare provided, in which a conjugate of general formula VI is formed fromthe sulfhydryl-containing compound and the conjugate is thenadministered to the mammal (for example, in an aqueous solution or anoral dosage unit).

Pursuant to yet another aspect of the present invention, there areprovided compounds of the general formula VA-S—S—CH₂—CR¹(NHCOR²)C(═O)R³  Vin which A is an aromatic activating residue (as hereinafter defined)and R¹, R² and R³ are as previously defined. The compounds of generalformula V are particularly useful in preparation of conjugates ofgeneral formula VI from sulfhydryl-containing compounds of generalformula PSH.

Pursuant to still another aspect of the present invention, there areprovided method for forming conjugates of general formula VI fromsulfhydryl-containing compounds of general formula PSH, which comprisesreacting a compound of general formula PSH with a compound of generalformula V. The reaction is typically carried out with an excess (e.g., atwo-fold to a ten-fold excess) of the compound of general formula V fora period of time of about 1 hour to about 24 hours at a temperature ofabout 4° C. to about 37° C. in a suitable aqueous buffer solution (e.g.,phosphate, bicarbonate and borate buffers). Preferably, the reaction iscarried out in bicarbonate buffer, pH 8.

Pursuant to another aspect of the present invention, there are providedcompounds of the general formula IIIA-S—S—CH₂—CR¹(NH₂)C(═O)R^(3′)  IIIin which R^(3′) is —OH or an amino acid chain comprising one or twoamino acids and terminating in —CO₂H and A and R¹ are as previouslydefined. The compounds of general formula III are useful in preparingthe compounds of general formula V. The compounds of general formula IIIare suitably prepared by reacting a compound of general formula IIH—S—CH₂—CR¹(NH₂)C(═O)R^(3′)  IIwith a compound of general formula A-S—S-A or A-S—S-A′, in which A′ isdifferent from A and is an aromatic activating residue. These reactantsare either commercially available [e.g., 2,2′-dithiopyridine and5,5′-dithiobis(2-nitrobenzoic acid)]or may be prepared by routinesynthetic procedures well known to those skilled in the art.

Pursuant to still another aspect of the present invention, there areprovided methods for preparation of compounds of general formula V inwhich R² is a lipid group, wherein a compound of general formula III isreacted with an activated lipid group of general formula X—O₂C—B orX—OC—B, in which X is a lipid-activating group (as hereinafter defined)and B is a lipid group (as hereinafter defined). Compounds of generalformula X—O₂C—B or X—OC—B may be readily prepared in a manner known perse.

For preparation of a compound of general formula III, in an exemplaryprocedure generally equal molar quantities of a compound of generalformula II and a compound of formula A-S—S-A or A-S—S-A′ may suitably bemixed in a polar organic solvent (e.g., ethanol). The product of generalformula III may then suitably be isolated by crystallization from anonpolar organic solvent (e.g., benzene). Of course, other suitableprocedures would also be evident to those working in the field.

For preparation of X—O₂C—B or X—OC—B, a fatty acid may for example bereacted with: (a) N-hydroxysuccinimide and a carbodiimide reagent toform an H-hydroxysuccinimidyl active ester; (b) trifluoroaceticanhydride to form a fatty acid anhydride; or (c) thionyl chloride toform a fatty acid chloride. Alternative procedures may also suitably beemployed to introduce these or other lipid-activating groups.

For purposes of the present invention, the term “lipid-containingmoiety” refers to either a lipid group per se or a hydrocarbon-basedgroup (in particular, one or more amino acids) comprising a lipid group.By the term “lipid group” is meant a hydrophobic substituent comprisingabout 4 to about 26 carbon atoms, preferably about 5 to about 19 carbonatoms. Suitable lipid groups include, but are not limited to, thefollowing: palmityl (C₁₅H₃₁); oleyl (C₁₅H₂₉); stearyl (C₁₇H₃₅); cholate;and deoxycholate.

By “aromatic activating residue” is meant a moiety which serves to makethe disulfide group of the compounds of general formula V more labile tothe displacement reaction with the sulfhydryl-containing compounds ofgeneral formula PSH (and thus, serves as a good leaving group). Apresently preferred aromatic activating group is 2-pyridyl; othersuitable aromatic activating groups include 4-nitrophenyl.

The term “lipid-activating group” refers for purposes of the presentinvention to a moiety which renders a carboxylipid group to which it isattached reactive with a compound of general formula III. A presentlypreferred lipid-activating group is N-hydroxysuccinimidyl ester; othersuitable lipid-activating groups include acid chloride and acidanhydride.

While the present invention contemplates the preparation and use ofconjugates of general formula VI comprising a wide range of compoundscontaining sulfhydryl groups, it is particularly advantageous to employthe methods and compositions of the present invention for preparation ofconjugates comprising biopolymers. Biopolymers of interest includepeptides, proteins, and oligonucleotides (as hereinafter defined). Aswould be readily apparent to those working in the field. biopolymers orthiolated biopolymers containing sulfhydryl groups may comprise aplurality of moieties corresponding in structure to the conjugates ofgeneral formula VI (i.e., groups having the structure of the compoundsof general formula VI minus the moiety P).

For purposes of the present invention, the term “peptide” refers toamino acid chains comprising two to 50 amino acids and the term“protein” to amino acid chains comprising more than 50 amino acids. Theproteins and peptides may be isolated from natural sources or preparedby means well known in the art, such as recombinant DNA technology orsolid-state synthesis. It is contemplated that the peptides and proteinsused in accordance with the present invention may comprise onlynaturally-occurring L-amino acids, combinations of L-amino acids andother amino acids (including R-amino acids and modified amino acids), oronly amino acids other than L-amino acids. In order to form a conjugateof general formula I, the peptide or protein must bear at least onereactive thiol group. In many cases, the peptide or protein containscysteine residues (an amino acid comprising a thiol group). A peptide orprotein which does not contain a thiol group may be modified byprocedures well known per se to those working in the field; inparticular, well known thiolating agents [e.g.,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and 2-iminothiolane(Traut's reagent)] may be routinely employed for this purpose.

The term “oligonucleotide” refers to chains comprising two or morenaturally-occurring or modified nucleic acids, for examplenaturally-occurring or recombinant deoxyribonucleic acids (DNA) andribonucleic acid (RNA) sequences. For formation of a conjugate inaccordance with the present invention, the oligonucleotide must bemodified by thiolating reactions so as to contain a sulfhydryl group forlinking with the lipid-containing moiety. Such modifications may beroutinely carried out in a manner known per se. For example, anoligonucleotide may be coupled to cystamine using carbodiimide andsubsequently reduced by dithiothreitol to generate a free sulfhydrylgroup.

In one preferred class of compounds of general formula VI, R¹ ishydrogen, R² is a lipid moiety and R³ is —OH. This type of conjugate issuitably derived from cysteine. In another preferred class of conjugatein accordance with the present invention, R¹ is hydrogen, R² is—CH₂CH₂CH(NH₂)CO₂H or —CH₂CH₂CH(NHCO-lipid)CO-lipid and R³ is —NHCH₂CO₂Hor —NHCH₂CO-lipid in which at least one of R² and R³ comprises a lipidmoiety. This type of conjugate is suitably derived from glutathione.

The synthesis of an exemplary compound of general formula VI (in which Pis a protein) is illustrated in Scheme I. Of course, as would be readilyappreciated by those skilled in the art, a variety of alternativesynthetic schemes could also readily be developed.

The fatty acid conjugates of the present invention are soluble in mostbuffer solutions in which proteins and peptides are soluble. Inparticular, any free carboxylic acid groups are charged at neutral pHand therefore improve the solubility of the conjugates. This greatlyfacilitates the formulation of the conjugates with suitablepharmaceutically-acceptable carriers or adjuvants for administration ofthe proteins or peptides to a patient by oral or other routes.

It is a particular advantage in accordance with the present inventionthat the disulfide linkage between the fatty acid moiety and the peptideor protein may readily be reduced. Therefore, the active peptide orprotein molecules are released in intact form inside the target tissuesor cells. Furthermore, the fatty acid moiety of the conjugates comprisesonly amino acids and lipid molecules which are not toxic to mammals, inparticular humans.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe invention as defined in the claims appended hereto.

EXAMPLES Example 1 Synthesis of N-palmityl-2-pyridyldithiocysteine(Pal-PDC)

An ice-cold solution of L-cysteine (I) (3.0 g) in ethanol (50 ml) wasadded dropwise to a stirred solution of 2,2-dithiopyridine (II) (7.5 g)in ethanol (30 ml), and the reaction was allowed to proceed at 25° C.for 18 hr. The solution was centrifuged in order to remove anyprecipitate, and the supernatant was reduced in volume to 40 ml using arotary evaporator. Subsequently, the reaction mixture was added dropwiseto 400 ml of ice-cold benzene. PDC (III), which crystallized in benzene,was isolated by filtration, redissolved in 40 ml of ethanol, and thenrecrystallized in 400 ml of ice-cold benzene as described above. Therecrystallized product was isolated by filtration, dried under vacuumovernight, and finally stored at −20° C. in a desiccator.

PDC (100 mg) (III) was dissolved in 5 ml of DMF and mixed with 100 μl oftriethylamine, and the resultant suspension was reacted with theN-hydroxysuccinimide ester of palmitic acid (IV) (250 mg) in DMF (5 ml)at 25° C. for 24 hr, during which time the suspension turned clear. Thissolution was diluted with 40 ml of ice-cold water, pH 3.0, and theprecipitate, which contained Pal-PDC (V) and palmitic acid, was isolatedby centrifugation at 10000 rpm for 30 min. Pal-PDC (V) was separatedfrom palmitic acid by suspension of the precipitate in water, pH 7.0,which dissolved Pal-PDC (V), but not palmitic acid. Pal-PDC (V) waspurified further using two more steps of acid precipitation as describedabove.

Example 2 Synthesis of Conjugates

Unless otherwise stated, all the final reagents used in the conjugationsteps (Pal-PDC and PDC) were analyzed using silica-coated thin layerchromatography (TLC) plates containing fluorescent indicators. Theseplates were not activated by heating prior to any of the analyses. Forthe routine analysis of the reagents synthesized, 5 μl of a ethanolicsolution containing the reagent (5 mg/ml) was applied to the plates.Subsequently, the plates were developed in solvent chambers,equilibrated with the mobile phase. Once the solvent front had travelleda sufficient distance, the plates were removed, dried, and studied undera UV-lamp. Positions of the spots were marked on the plates immediately,and a drawing of the plate and the spots was made. The Rf value for eachspot visualized was calculated and recorded. The composition of themobile phases used in the analyses were adjusted to provide optimumseparation of the reagent-spots.

For purposes of illustration, conjugates of BBI were synthesized. BBI isa hydrophilic protein which has low uptake into cells and is not orallybioavailable. In addition, BBI is stable in the GI tract and resistsdegradation by the mammalian proteases in the gut [Yavelow, J. et al.(1983) Cancer. Res. 43, 2454s–2459s]. The use of BBI for chemopreventioncan be accepted only if an orally absorbable form of BBI can bedeveloped.

BBI (20 mg) was dissolved in 1 ml of a sodium bicarbonate solution (0.3M, pH 8.0) and reacted with SPDP (5 mg/100 μl of DMF) for 2 hr at 25° C.After purification of BBI-PDP using Sephadex® G50 gel-filtrationchromatography, the PDP-derivatization of BBI was estimated by measuringthe release of the thiopyridine moiety after reduction of BBI-PDP withdithiothreitol (DTT). Using this procedure, approximately 4 amino groupsper BBI molecule were modified with SPDP. The level of derivatization ofBBI could be controlled by adjusting the pH of the reaction buffer; themodification of BBI could be adjusted from one amine group per BBImolecule when the reaction was carried out at pH 7, to 4.5 amine groupsmodified when the reaction was carried out at pH 8.5.

BBI-PDP (20 mg) in PBS (1 ml, pH 5.0) was reduced with DTT (25 mM) for30 min and subsequently eluted from a Sephadex® G50 column. Thesulfhydryl-containing BBI fractions, which eluted at the column voidvolume, were identified using Elman's reagent, and then reacted with a3-fold excess (per sulfhydryl group on BBI) of Pal-PDC (V) in PBS, pH7.0, for 16 hrs at 4° C. The reaction mixture was then acidified to pH3.0 using HCI (1M) and left on ice for 30 min. The supernatant wasanalyzed separately using a Sephadex G25 gel-filtration column. Theprecipitate, which contained the palmityl disulfide conjugate of BBI,BBIssPal (VI), and the excess reagent, was isolated by centrifugation,dissolved in DMF (2 ml), and eluted from a Sephadex® LH20 column usingDMF. BBIssPal (VI) fractions, which eluted at column void volume, wereisolated, dialyzed 3 times against 500 volumes of water, and thenlyophilized. The yield of the conjugate using this procedure wasapproximately 80% (by weight). The conjugation of Pal-PDC to BBI wasconfirmed and quantitated after the conjugation of [3H]-labeled Pal-PDC(V) to BBI using identical conjugation conditions as the ones describedabove. Also, using an identical procedure, the oleic acid conjugated BBI(BBIssOleic) was synthesized.

Example 3 Transport of Conjugates

Human colon carcinoma cells (Caco-2) were detached from 25 cm² stockculture flasks using a 10 min incubation at 37° C. with 0.5 ml of atrypsin/EDTA solution (0.5% trypsin, 5.3 mM EDTA). The cells were thensuspended in 5 ml of Dulbecco's minimum essential medium, supplementedwith 15% fetal bovine serum (FBS), L-glutamine (1%), and essential aminoacids (1%), and counted using a coulter counter.

Suspended Caco-2 cells in 1.5 ml of medium were seeded into the apicalchamber of the transwells at a density of 0.5 million cells per insert.2.5 ml of the medium was then added to the basal chambers of eachtranswell. The cells were allowed to attach for 2 days withoutdisturbance and were then fed every other day until the experiments wereperformed. The cells were maintained for approximately 14–20 days priorto the experiments and were fed 24 hr before each experiment. The cellmonolayers developed a transepithelial electrical resistance (TEER) ofapproximately 500–600 Ωcm² within one week of the seeding and maintainedthis resistance for up to 21 days post-seeding.

Radioiodination of BBI and BBIssPal was carried out using thechloramine-T method [McConahey, P. C., and Dixon, F. J. (1980) Meth.Enzymol. 70, 221–247]. Confluent, 14-day old cell monolayers were washedonce with, and then incubated in, serum-free Dulbecco medium at 37° C.for 30 min. Subsequently, the incubation medium was replaced with serumfree medium containing ¹²⁵I-BBI (10 μg/ml), either as native-BBI or asBBIssPal or BBIssOleic, and the monolayers were incubated for a further60 min at 37° C. The monolayers were then washed three times withice-cold PBS, and then exposed to trypsin (0.5%, EDTA 5.3 mM) for 10 minat 37° C. The detached cells were transferred to tubes, isolated bycentrifugation, washed three times using ice-cold PBS, assayed foraccumulated radioactivity using a gamma counter, and finally assayed forcell protein using the published method [Lowry, O. H. et al. (1951) J.Biol. Chem. 193, 265–275].

In some experiments the uptake of reduced ¹²⁵I-BBIssPal into cells wasdetermined. ¹²⁵I-BBIssPal was reduced with DTT (50 mM) at 60° C. for 5min followed by a further 25 min at 37° C. In control experiments,¹²⁵I-BBIssPal was exposed in medium to the same temperatures withoutbeing exposed to DTT.

The uptake of ¹²⁵I-BBIssPal in the presence of BSA (fatty acid free) wasdetermined as follows. ¹²⁵I-BBIssPal was incubated with mediumcontaining 0.1% BSA for 30 min at 37° C. before being added to the cellmonolayers. In some uptake experiments, BSA was first mixed with a 3fold molar excess of palmitic acid, and then incubated with theconjugates prior to the experiments. In the experiments where the uptakeof ¹²⁵I-BBIssPal was determined in medium containing FBS, the conjugateswas simply added to the medium containing the required amount of FBS.

Confluent cell monolayers, 2 to 3 weeks old, and having a TEER value ofapproximately 500 Ωcm², were first incubated with Dulbecco's MEMcontaining 1% of FBS for 30 min at 37° C. Subsequently, the incubationmedium was removed, and the ¹²⁵I-BBI (10 μg/ml) conjugates in 1.5 ml ofthe medium was added to the apical chamber of the transwells. To thebasal chamber, 2.5 ml of the medium was added and the transwells wereincubated at 37° C. At predetermined times, the entire basal chambermedium (2.5 ml) from each transwell was removed and counted forradioactivity using a gamma counter. In each experiment, typically sevensamples were taken at 1, 2, 3, 4, 5, 6 and 24 hr post-incubation. Afterthe 24 hr samples were taken, the cell monolayers were rinsed threetimes with ice-cold PBS, cut out of the inserts, and counted foraccumulated radioactivity using a gamma counter.

The integrity of the ¹²⁵I-BBI conjugates transported across themonolayers was studied using Sephadex G50 gel-filtration chromatography.Briefly, after the basal medium was sampled at 24 hr, 1.0 ml of themedium was centrifuged at 2000 rpm and then eluted from a G50 column (10ml) using PBS; 1 ml fractions were collected and the fraction-associatedradioactivity was determined using a gamma counter. Intact conjugateseluted at column void volume and fragments smaller than 1 kDa wereeluted at or above the column volume.

The results of the uptake of ¹²⁵I-BBI, either as the free protein or inconjugated form to palmitic acid, in the presence of different amountsof added FBS are shown in Table 1. When the conjugates were incubatedwith the cells in serum-free medium, the uptake of BBIssPal wasapproximately 140-fold higher than that of BBI. In the presence ofmedium containing 1% FBS, the internalization of BBIssPal was increasedby 35-fold over that of BBI. Increasing the serum concentration furtherto 10%, caused a further decrease in the uptake of BBIssPal into thecells to only a 10-fold higher level than that of native-BBI. Theinternalization of BBIssPal into Caco-2 cells was reduced drastically inthe presence of serum to 14% and 2.3% of that of the serum-free valuesfor 1% and 10% FBS containing media, respectively.

TABLE 1 Uptake (ng BBI/mg of cell protein)/hr serum free 1% FBS 10% FBSBBI 3.9 ± 0.19 2.2 ± 0.17 1.3 ± 0.02 BBIssPal 540.0 ± 24.13  78.5 ±3.41  12.9 ± 0.02 The cell monolayers were incubated with 125I-labeled conjugates at 10μg/ml for 60 min at 37° C. The results presented are the average ofthree monolayers ± SEM. The uptake experiments were carried out inDulbecco medium, in the presence and absence of added FBS.

Since the BBIssPal uptake into the cells was believed to be mediated bythe palmitic acid ligands on the conjugate, the uptake of ¹²⁵I-BBIssPalinto Caco-2 cells before and after reduction with DTT was studied. Sincethe presence of serum in the incubation medium had an inhibiting effecton the uptake of the conjugates into the cells, the uptake was studiedin serum-free medium. The results are shown in Table 2. The uptake ofuntreated ¹²⁵I-BBIssPal into the cells was 80-fold higher than that of¹²⁵-BBI. The exposure of ¹²⁵I-BBI to DTT did not cause a reduction inthe uptake. In contrast, the reduction of ¹²⁵I-BBIssPal with DTT reducedthe uptake of the conjugate in to the cells by approximately 80%. Thereduction of BBIssPal with DTT causes the detachment of the palmiticacid from the conjugate. Hence, the uptake of ¹²⁵I-BBIssPal was mediatedby the hydrophobic palmitic acid ligand.

TABLE 2 Uptake (ng BBI/mg of cell protein)/hr Untreated DTT-treated BBI4.8 ± 0.00 5.2 ± 0.00 BBIssPal 381.7 ± 0.03  46.5 ± 0.00 The cell uptake of ¹²⁵I-BBI, either as the native protein or as BBIssPalwas determined before and after reduction with DTT (50 mM) for 5 min at60° C. and 25 min at 37° C.

Bovine serum albumin (BSA) is known to be a carrier of fatty acids invivo and contain hydrophobic regions which can tightly bind fatty acids.Since the uptake of ¹²⁵I-BBIssPal was reduced in the presence of serum,the possibility that BBIssPal bound to BSA present in FBS wasinvestigated. The cell uptake of ¹²⁵I-BBIssPal and ¹²⁵I-BBI in thepresence of medium containing fat-free BSA or fatty acid-loaded BSA wasstudied, and the results are shown in Table 3. In the presence ofBSA-free medium, the uptake of ¹²⁵I-BBIssPal into the cells was 80-foldhigher than that of BBI, as was expected from the results obtained inthe previous experiments. When defatted-BSA (fatty acid-free BSA) (0.1%)was present in the medium, the uptake of ¹²⁵I-BBIssPal was reduced by82%, whereas the uptake of ¹²⁵I-BBI was not affected. In the presence offatty acid-loaded BSA (0.1%), which was produced by spiking fat-free BSAwith a 3-molar excess of palmitic acid, the uptake of ¹²⁵I-BBI was againnot affected. Therefore, ¹²⁵I-BBIssPal binds strongly to BSA and thisbinding is dependent on the number of fatty acids already bound to BSA.

TABLE 3 Uptake (ng BBI/mg of cell protein)/hr serum free BSA BSA/FA BBI4.8 ± 0.00 4.8 ± 0.00 3.9 ± 0.00 BBIssPal 380.0 ± 0.03  69.7 ± 0.00 258.9 ± 0.00 

The uptake experiments were carried out in Dulbecco medium, in thepresence and absence of added fatty acid-free BSA (BSA) or fattyacid-loaded BSA (BSA/FA). The results of studies of the uptake of¹²⁵I-BBI, either as the native-BBI or in conjugated form to palmitic oroleic acid, in Caco-2 cells in the presence of serum-free medium weredetermined as the average ng of BBI internalized ± SEM, n=3. The uptakeof ¹²⁵I-BBIssPal into the cells was approximately 100-fold higher thanthat of ¹²⁵I-BBI. Similarly, the uptake of ¹²⁵I-BBIssOleic into thecells was about 108-fold higher than ¹²⁵BBI. The difference between theuptake of ¹²⁵I-BBIssPal and ¹²⁵I-BBIssOleic were not significant.

Example 4 Biodistribution Assays

Female CF-1 mice, 2 to 3 weeks old, weighing 20–25 g each, with freeaccess to food and water prior to the experiments, were used for theanimal experiments. ¹²⁵I-BBI (3 mg/kg), as native-BBI or as BBIssPal orBBIssOleic conjugate, was administered to the animals via the tail vein.At 0.5, 3, and 24 hr post-injection, 3 animals from each experimentgroup were sacrificed and their blood (200 μl), the kidneys, the lungs,and the liver were removed, rinsed in ice-cold PBS, and assayed foraccumulated radioactivity. The weights of the organs were recorded andused to adjust the concentration of the conjugates in the organs.

In the ip-biodistribution studies, ¹²⁵I-BBI (3 mg/kg), either as thenative-BBI or as BBIssPal, was administered into the lower left quadrantof the abdominal cavity of each animal. The animals were then treated inthe manner described for the iv.-biodistribution studies.

The results of the biodistribution of BBI and BBIssPal followingiv-administration were determined as the % dose accumulated per g organ± SEM. The results indicated that while BBI was rapidly excreted fromthe body without attaining high blood levels, BBIssPal was accumulatedin the blood at a relatively high level and was apparently more slowlyremoved from the circulation. The kidney biodistribution resultsindicated that while BBI was rapidly accumulated in the kidneys,BBIssPal was not. The liver accumulation of BBIssPal was approximately5-fold higher than that of BBI, and BBIssPal levels remained high in theliver even at 24 hr post-injection. The lung accumulation of BBIssPalwas also approximately 2-fold higher than that of BBI, but this resultmay have been caused by the residual blood present in the organ afterits excision. Clearly, BBIssPal was retained longer and at a higherlevel in the blood and the liver. On the other hand, the kidneyclearance of BBIssPal was about 4-fold lower than native-BBI.

The iv-biodistribution of BBI and BBIssOleic were also studied in CF-1mice. The results were determined as the % dose accumulated per g of theorgan ± SEM, n=3, at 0.5, 3 and 24 hr. The biodistribution of BBIssOleicwas very similar to BBIssPal. As was observed for BBIssPal, BBIssOleichad higher blood levels than BBI and was apparently more slowly clearedfrom the circulation. The blood levels of BBIssOleic were about 4-foldhigher than those of BBI at the same time points. The kidney clearanceof BBIssOleic was approximately 4-fold lower, and the liver accumulationapproximately 4-fold higher than native-BBI. The retention of BBIssOleicin the liver was prolonged, with significant levels of the conjugatepresent in the liver even at 24 hr post-injection. The lung levels ofBBIssOleic were about 2-fold higher than native-BBI levels, but thehigher residual blood in the lungs could account for this observation.

The ip-biodistribution of ¹²⁵I-BBIssPal in CF-1 mice was determined asthe average % dose accumulation per organ ± range at 0.5 hr, 3 hr or 24hr post-injection. The kidney accumulation of ¹²⁵I-BBIssPal was 4-foldlower than that of native ¹²⁵I-BBI for the 0.5 and 3 hr time points. At24 hr, ¹²⁵I-BBIssPal levels were higher in the kidneys than ¹²⁵I-BBI.The blood level of ¹²⁵I-BBIssPal was similar to that of ¹²⁵I-BBI at 0.5hr, 1.5 -fold higher than BBI at 3 hr, and approximately 3-fold higherthan BBI at 24 hr. The liver accumulation of ¹²⁵I-BBIssPal was 1.5-foldhigher than ¹²⁵I-BBI at 0.5 hr, 2.5-fold higher at 3 hr, and about4-fold higher at 24 hr. Relatively large amounts of ¹²⁵I-BBIssPal werepresent in the liver and the kidneys at 24 hr.

Example 5 In Vitro Transformation Studies

Transformation assays were carried out using C3H 10T1/2(clone 8) cellsaccording to the published recommendations [Reznikoff, C. A. et al.(1973) Cancer. Res. 33, 3239–3249; Reznikoff, C. A. et al. (1973)Cancer. Res. 33, 3231–3238]. Stock cultures of mycoplasma-free cellswere maintained by passing 50,000 cells per 75 cm² flask every sevendays. Using this schedule, the cells were always passed approximately 2days before reaching confluence. The stock culture was grown in Eagle'sbasal medium supplemented with 10% FBS, penicillin (100 units), andstreptomycin (100 μg) and used for the transformation assays at passagesof 9 to 14. The cells were passed by treating the stock cells withtrypsin (0.1%) in PBS for 5 min and quenching the trypsin with 5 ml ofthe medium. This procedure was adapted to minimize spontaneoustransformation in the stock cultures and maximize the plating efficiencyin the petri dishes. The FBS stock used in the cultures was pre-screenedto ensure that the serum was able to support the expression and thegrowth of the transformed cells.

For the transformation assays, C3H 10T1/2 cells (1000/dish) were seededinto 60 mm petri dishes and allowed to grow in a humidified 5% CO₂atmosphere in Eagle's basal medium, supplemented with 10% FBS,penicillin (100 units), and streptomycin (100 μg), for 24 hr.Subsequently, the cells were initiated by treatment with 25 μl of the3-methylcholanthrene (MCA) in acetone stock solution (0.25 mg/ml) to afinal concentration of 1 μg/ml of MCA (5 μg/5 ml). The cells wereallowed to grow in the presence of the carcinogen or solvent for 24 hr,and the medium in each dish was then replaced with fresh mediumcontaining no carcinogen or solvent. The medium in the dishes wasreplaced twice per week for the first two weeks of the assay, andthereafter once a week for the remainder four weeks of the assay. In theexperiments designed to determine the transformation inhibitory activityof the conjugates, the cells were maintained in the medium containingthe conjugates (1 μg/ml) for the first three weeks of the assay;thereafter, the cells were maintained in medium containing no addedconjugates.

Six weeks after the carcinogen treatment, the cells were inspected undera microscope for adherence to the culture dishes and were washed withPBS and then fixed in 100% methanol. The fixed monolayers were thenstained with Giemsa stain. 20 dishes per group were treated in eachexperiment. In addition to the test groups, all the transformationassays contained at least three other groups: negative control (nottreated with carcinogen or solvent), acetone control (treated with 25 μlof acetone), and positive control [treated with MCA (1 μg/ml) in 25 μlof acetone]. The transformed foci (>3 mm in diameter) in the plates werestudied under a microscope and classified according to publishedguidelines as types I, II, or III [Landolph, J. R. (1985) Transfomatinassay of established cell lines: Mechanism and Application (ed.Kakunaga, T., and Yamasaki, H.) IARC Scientific Publications, Lyon,France pp. 185–201]. Briefly, type III foci were dense, multilayered,basophilic, areas of cell growth which stained to a deep blue color withGiemsa and had rough criss-crossed edges. Type II foci were also dense,multilayered, areas of cell growth, but were stained to a purple colorwith Giemsa and had smoother, more defined edges compared to Type IIIfoci. Type I foci were not scored in the assay.

The plating efficiency (PE) of the cells was also studied in conjunctionwith each of the transformation assays. To determine the PE of the cellsin the different treatment groups, cells (200 cells/dish) were seededinto three 60-mm petri dishes per experiment group and treated in theidentical manner as the transformation assay cells. The cells in theseassays were terminated at 10 days, fixed with 100% methanol, and stainedwith giemsa; the colonies of 50 cells or more visible under a microscopewere then counted. The plating efficiency is defined as the (number ofcolonies/number of cells seeded)*100.

The in vitro anti-transformation activity of BBI, BBIssPal, andBBIssOleic is shown in Table 4. BBI, either as the free protein or inconjugated form to palmitic or oleic acid, was added to the cultures at1.0 μg/ml for the first three weeks of the transformation assay periodstarting immediately after the MCA treatment. MCA-treated cells wereexposed to 3-methylcholanthrene, dissolved in 25 /μl of acetone, at aconcentration of 1 μg/ml for 24 hr. Acetone-treated cells were exposedto 25 μl of acetone for 24 hr only. The test groups were exposed to MCAfor 24 hr and then to the conjugates for the first three weeks of theassay. Untreated cells were exposed to neither MCA nor acetone.Statistical analysis (Chi-square): Group 4 vs 3, p<0.05; Group 5 vs 3,0.05<p<0.1; Group 6 vs 3, p<0.05. Control, untreated cells reachedconfluence in the dishes about 14-days post-seeding formed welladherent, contact-inhibited monolayers. These dishes contained notransformed foci at the end of the assay period. The acetone treatedcells also reached confluence and formed well-adherent monolayers 14days post-seeding and contained no transformed foci. The MCA-treateddishes, however, contained morphologically transformed foci: 6 out ofthe 19 dishes scored contained type III foci. The BBI-treated groupcontained no transformed foci, indicating that BBI could preventMCA-induced transformation in these cells. The BBIssPal-treated cellscontained one type II focus out of the 20 dishes scored in the assay.The BBIssOleic treated cells contained no transformed foci. The PE ofall the groups in this assay was between 20% to 25%. As demonstrated inTable 4, both BBIssPal and BBIssOleic retained the original biologicalactivity of BBI.

TABLE 4 No. of dishes Fraction with of dishes transformed containingPlating foci/No. of transformed Treatment Group Efficiency (%) dishesfoci 1. Controls - untreated 23 ± 1.5 0/20 0 2. Negative controls - 22 ±2.0 0/20 0 acetone treated 3. Positive controls - 21 ± 3.0 6/19 0.32MCA-treated 4. Test-MCA 24 ± 2.0 0/20 0 treated + BBI 5. Test-MCA- 23 ±3.0 1/20 0.05 treated + BBIssPal 6. Test-MCA- 24 ± 3.5 0/20 0 treated +BBIssOleic

Example 6 Transport of Single- and Multiple-Conjugates

Studies on transport of apical membrane-bound ¹²⁵I-BBIssPal were carriedout using transwells and six-well plates. In the six-well plateexperiments, ¹²⁵I-BBI or ¹²⁵I-BBIssPal (10 μg/ml) was incubated withCaco-2 cells in serum-free medium for 1 hr at 37° C. Subsequently, thecells were rinsed three times with ice-cold PBS and then divided intotwo groups. In the first group the internalization of the conjugates wasdetermined after the trypsinization and isolation of the cells. In thesecond group, the cells were reincubated with serum-free medium and therelease of the conjugates from the cells was chased for 24 hr; mediumwas removed at hourly intervals and counted for radioactivity. At theend of the chase period, the cells were trypsinized, isolated, andcounted for accumulated radioactivity. The total counts in eachexperiments (medium+cell cpms) were determined, and the % of the totalcounts released at different times was determined.

In the transwell experiments, the conjugates were incubated with theapical side of the cells for 1 hr at 37° C. The transwells were thenrinsed three times with ice-cold PBS and then reincubated with serumfree medium. The release of the conjugates into the apical and the basalmedium was chased for 24 hr by counting the entire basal or the apicalmedium at different times. The total counts obtained at the end of thechase period (transwells+media counts were added, and the release of theconjugates (% of total) at different times was calculated. To ensurethat the counts obtained in the transwells at 24 hr were due to thepresence of the conjugates in the cells and not non-specific binding tothe plastic, the transwells were exposed to trypsin for 10 min, rinsedthree times with ice-cold pbs, and subsequently counted for accumulatedradioactivity.

BBI was modified with 2 or 4 palmitic acids, and the transport wasdetermined in transwells. The cumulative transport of BBI, BBI modifiedwith 4 palmitic acids, and BBI modified with 2 palmitic acids in Caco-2monolayers were determined as BBI (ng/monolayer) ± SEM, n=3. The orderof the transport extent was BBIssPal(4)>BBI>BBIssPal(2). The results ofthe internalization of the conjugates into the same cells weredetermined as the ng of BBI internalized per monolayer. As expected,BBIssPal(4) had the highest uptake into the cells, followed byBBIssPal(2) and BBI. The basal media obtained at 24 hr from thetranswells was analyzed using a G50 column. As had been observed before,neither BBI nor BBIssPal(4) was transcytosed across the monolayers.However, a small, but significant, amount of the basal media ofBBIssPal(2) consisted of intact conjugate. This quantity consisted ofbetween about 10 and about 20% of the total radioactivity present in thebasal medium.

Example 7 Skin Absorption of BBIssPal

Freshly-prepared skins from hairless mice were mounted on small rings.To each mounted skin, a 5 μl sample of ¹²⁵I-labeled BBI or BBIssPal at aconcentration of 0.5 mg/ml was applied to an area of 0.38 cm². Twopieces of skin were used per treatment. The skins were kept at roomtemperature (23° C.) in a humidified environment. After 30 minutes, thesurface of the skins was first rinsed carefully with PBS; subsequently,the skins were unmounted and soaked twice in 100 ml of PBS. The skinswere then blotted with filter papers and counted in a gamma counter. Theamount of BBI retained on the skins was calculated using the specificradioactivity of the labeled BBI or BBIssPal. The absorption of BBI andBBIssPal into the mouse skins was 0.14 and 1.6 μg/cm², respectively.This demonstrates that a more than 10-fold increase of BBI absorptioninto the skin was achieved when the polypeptide was modified usingPal-PDC.

Example 8 Synthesis of Palmitylated Horseradish Peroxidase (HRPssPal)

Ten milligrams of horseradish peroxidase (molecular weight 40,000; SigmaP 8375) in 0.5 ml of PBS was mixed with 2 ml of SPDP in 0.1 ml DMF at25° C. for two hours. The reaction was terminated by dilution with 0.5ml PBS, and dialyzed in 500 ml of PBS at 4° C. After 24 hours, thesolution in the dialysis tube was removed, reduced by the addition of 50μl of 1 M DTT, and separated by using a Sephadex G-50 column. Fractionsat the void volume of the column were pooled and mixed with a 10-foldmolar excess of Pal-PDC in borate buffer, pH 9.6 at 25° C. for 4 hours.The reaction mixture was then dialyzed exhaustively at 4° C. for 3 days,and the final product was estimated to contain 10 palmitic acid residuesper molecule of HRP. The HRP molecules retained approximately 20% of theoriginal enzyme activity.

Example 9 Cellular Uptake of HRPssPal

Confluent monolayers of mouse fibroblasts L929 cells in 6-well culturecluster plates were incubated in serum-free medium with 30 μg/ml of HRP,either as the native form or as the palmitic acid conjugate (HRPssPal).After 1 hour at 37° C., monolayers were washed three times with PBS andthen dissolved in 1 ml of 0.05% of Triton-X100. Cell-associated HRP wasdetermined by measuring the enzymatic activity in each cell extract andthe results converted to ng HRP per cell monolayer. Results indicatedthat cellular uptakes of HRP and HRPssPal were 7 and 229 ng HRP per cellmonolayer, respectively. Therefore, a 30-fold increase in cell uptakewas achieved by modification of HRP with Pal-PDC.

Example 10 Lipidization of Oligonucleotides

An antisense 21mer oligonucleotide which is complementary to the mRNA ofmonoamine oxidase B is thiolated using the following procedure. Theoligonucleotide is mixed with a two-fold molar excess of cystamine inthe presence of a water-soluble carbodiimide reagent, EDC. The mixtureis maintained at 25° C. for 2 hours and the a two-fold molar excess tocystamine of DTT is added to reduce disulfide bonds.

After separating the oligonucleotide from free cystamine and DTT using aSepahdex G-25 column, a small amount of the thiolated oligonucleotide isreacted with Ellman's reagent and the concentration of sulfhydryl groupsdetermined using the absorbance at 412 nm (assuming an ∈ of 1.36×10⁴M⁻¹). Subsequently, the number of sulfhydryl groups per oligonucleotidemolecule is determined. The thiolated oligonucleotide is mixed inbicarbonate buffer, pH 8, with Pal-PDC in two-fold molar excess to thenumber of sulfhydryl groups in the oligonucleotide. The palmitylatedoligonucleotide is purified using a Sephadex G-25 column.

From the foregoing description, one skilled in the art can readilyascertain the essential characteristics of the invention and, withoutdeparting from the spirit and scope thereof, can adapt the invention tovarious usages and conditions. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient, and any specific terms employed herein are intended in adescriptive sense and not for purposes of limitation.

1. A method for prolonging blood and tissue retention of asulfhydrylgroup containing compound selected from the group consistingof peptides, proteins and oligonucleotides into mammalian cells, saidmethod comprising: forming from the sulfhydryl-containing compound acompound of general formula VI

in which P is selected from the group consisting of peptides, proteinsand oligonucleotides; R¹ is hydrogen, lower alkyl or aryl; R² isselected from the group consisting of a lipid, —CH₂CH₂CH(NH₂)CO₂H and—CH₂CH₂CH(NHCO-lipid)CO-lipid; and R³ is —OH, a lipid or an amino acidchain comprising one or 2 amino acids and terminating in —CO₂H or —COR²;and administering the compound of general formula VI to the cells;wherein said lipid is a hydrophobic substituent consisting of 4 to 26carbon atoms and said lipid together with the attached carbonyl is afatty acid acyl group.
 2. A method according to claim 1, wherein R¹ ishydrogen, R² is a lipid group and R³ is —OH.
 3. A method according toclaim 1, wherein R¹ is hydrogen, R² is —CH₂CH₂CH(NH₂)CO₂H or—CH₂CH₂CH(NHCO-lipid)CO-lipid and R³ is —NHCH₂CO₂H or —NHCH₂CO-lipid inwhich at least one of R² and R³ comprises a lipid group.